1. Field of the Invention
The present invention relates generally to the detection and measurement of transmembrane potentials using an N,N,N′-trialkyl thiobarbituric acid-derived polymethine oxonol. In particular, the present invention is directed to compositions and optical methods for determining transmembrane potentials across the plasma membrane of biological cells using a slightly hydrophobic N,N,N′-trialkyl thiobarbituric acid-derived polymethine oxonols. The method comprises a slightly hydrophobic N,N,N′-trialkyl thiobarbituric acid-derived polymethine oxonol anion capable of redistributing from a first face of the membrane to a second face of the membrane in response to changes in the potential of the membrane. In one aspect the method is used to identify compounds which modulate membrane potentials in biological membranes.
2. Background of the Art
The plasma membrane of a cell typically has a transmembrane potential of approximately −70 mV (negative inside) as a consequence of K+, Na+ and Cl− concentration gradients that are maintained by active transport processes. Increases and decreases in membrane potential (referred to as membrane hyperpolarization and depolarization, respectively) play a central role in many physiological processes, including nerve-impulse propagation, muscle contraction, cell signaling and ion-channel gating [Shapiro H M. “Cell membrane potential analysis.” Methods Cell Biol 41, 121-133 (1994); Baxter D F, Kirk M, Garcia A F, Raimondi A, Holmqvist M H, Flint K K, Bojanic D, Distefano P S, Curtis R, Xie Y. “A novel membrane potential-sensitive fluorescent dye improves cell-based assays for ion channels.” J Biomol Screen 7, 79 (2002); Falconer M, Smith F, Surah-Narwal S, Congrave G, Liu Z, Hayater P, Ciaramella G, Keighley W, Haddock P, Waldron G, Sewing A. “High-throughput screening for ion channel modulators” J Biomol Screen 7, 460 (2002)]. In general, there are two distinct methods to measure cell membranes, (a) direct electrical measurement of cell membrane potentials, e.g, the so-called ‘Patch Clamping’ technique, and (b) indirect optical sensing of membrane potentials using a membrane potential-sensitive dye as an indicator. Fluorescence detection and imaging of cellular electrical activity is a technique of great importance [Grinvald, A., Frostig, R. D., Lieke, E., and Hildesheim, R. “Optical imaging of neuronal activity.” Physiol. Rev. 68, 1285-1366 (1988); Salzberg, B. M. “Optical recording of electrical activity in neurons using molecular probes.” In Current Methods in Cellular Neurobiology. J. L. Barker (editor) Wiley, New York. 1983, pp 139-187; Cohen, L. B. and S. Lesher. “Optical monitoring of membrane potential: methods of multisite optical measurement.” In Optical Methods in Cell Physiology. P. de Weer and B. M. Salzberg (editors), 1985, Wiley, New York. pp 71-99]. The optical method that uses a fluorescent indicator has steadily gained popularity in recent years due to its convenience, high throughput and improved sensitivity. Potentiometric probes are a critical factor in the optical measurement of membrane potentials. The existing potentiometric probes include the cationic or zwitterionic styryl dyes, the cationic carbocyanines and rhodamines, the anionic oxonols and hybrid oxonols and merocyanine 540. The class of dyes determines factors such as accumulation in cells, response mechanism and toxicity. The fluorescent indicators used in the optical measurement of membrane potential have been traditionally divided into two classes:
(1) Fast-Response Dyes:
These dyes are usually cell-impermeable and have fast response to changes in membrane potentials because they need little or no translocation. [Loew, L. M., “How to choose a potentiometric membrane probe”, In Spectroscopic Membrane Probes. CRC Press, Boca Raton L., 1988, pp 139-151; Loew, L. M., “Potentiometric membrane dyes”, In Fluorescent and Luminescent Probes for Biological Activity. W. T. Mason (editor), Academic Press, San Diego, 1993, pp 150-160]. However, they are insensitive because they sense the electric field with only a part of a unit charge moving less than the length of the molecule, which in turn is only a small fraction of the distance across the membrane. Furthermore, a significant fraction of the total dye signal comes from molecules that sit on irrelevant membranes or cells and that dilute the signal from the few correctly placed molecules.
(2) Slow-Response Dyes:
In contrast to the above-mentioned ‘fast-response’ dyes, these dyes are usually hydrophobic and cell-permeable. They are quite sensitive although they have a slow redistribution of permeant ionized dyes from the extracellular medium into the cell. The ratio of their concentrations between the inside and outside of the cell can change by up to the Nernstian limit of 10 fold for a 60 mV change in transmembrane potential. However, for the permeable ions to establish new equilibria, the dye ions must diffuse through unstirred layers in each aqueous phase and the low-dielectric-constant interior of the plasma membrane. These processes result in their slow responses to changes in membrane potentials. Moreover, such dyes distribute into all available hydrophobic binding sites indiscriminately. Therefore, selectivity between cell types is difficult. Additionally, any additions of hydrophobic proteins or reagents to the external solution, or changes in exposure to hydrophobic surfaces, are prone to cause artifacts.
In view of the above drawbacks of existing fluorescent dyes used in optical measurement of membrane potentials, improved methods and compositions are needed to detect small variations in transmembrane potentials with a rapid response and strong fluorescence signal, preferably on a millisecond to second timescale. Also urgently needed are methods and compositions less susceptible to the effects of changes in external solution composition, in particular, eliminating the serum effect. The critical factors to develop such membrane potential detection technologies are the effective design and synthesis and testing/screening of membrane potential-sensitive fluorescent dyes. This invention fulfills this and related needs.
The thiobarbituric acid-based oxonols, often referred to as “DiSBAC” dyes (in the case of symmetric thiobarbituric acid-derived polymethine oxonols) form a family of spectrally distinct potentiometric probes with excitation maxima covering most range of visible wavelengths. DiSBAC2(3) has been the most popular oxonol dye for membrane potential measurement [Plasek J, Sigler K. “Slow fluorescent indicators of membrane potential: a survey of different approaches to probe response analysis.” J Photochem Photobiol B 33, 101-124 (1996); Loew L M. “Characterization of Potentiometric Membrane Dyes.” Adv Chem Ser 235, 151 (1994); Loew, L. M., “How to choose a potentiometric membrane probe”, In Spectroscopic Membrane Probes. CRC Press, Boca Raton L., 1988, pp 139-151; Loew, L. M., “Potentiometric membrane dyes”, In Fluorescent and Luminescent Probes for Biological Activity. W. T. Mason (editor), Academic Press, San Diego, 1993, pp 150-160]. These dyes enter depolarized cells where they bind to intracellular proteins or membranes and exhibit enhanced fluorescence. Increased depolarization results in more influx of the anionic dye and thus an increase in fluorescence.
In general, DiSBAC dyes bearing longer alkyl chains had been proposed to have better properties for measuring membrane potentials [Loew L M., “Potentiometric Membrane Dyes”. In Fluorescent and Luminescent Probes for Biological Activity, Mason W T, 2.sup.nd Ed. 1999, pp 210-221; Gonzalez J E, Tsien R Y. “Improved indicators of cell membrane potential that use fluorescence resonance energy transfer.” Chem Biol 4, 269-277 (1997)]. Recently this hypothesis has been disputed by the fact that DiSBAC1(3) possess better properties for optical measurement of membrane potentials than DiSBAC2(3) (U.S. Patent Application 20030087332). In this invention, DiSBAC6(3) and DiSBAC0(3) are prepared to confirm the existing theories of designing effective fluorescent indicators for measuring membrane potentials. Neither of the compounds prove to be better fluorescent indicators for measuring membrane potentials than DiSBAC1(3) although DiSBAC0(3) or DiSBAC6(3) would have been a better fluorescent membrane potential indicator [than DiSBAC1(3)] according to U.S. Patent Application 20030087332 or according to the generally accepted hypothesis that more hydrophobic oxonols tend to be better fluorescent membrane potential indicators.
The existing thiobarbituric acid-derived polymethine oxonols used in optical measurement of membrane potentials are symmetric oxonols that are referred as ‘DiSBAC’ and have the four same alkyl groups on the two thiobarbituric acid moieties. In our previous disclosure (U.S. patent application Ser. No. 10/971,311) we discovered that the thiobarbituric acid-derived polymethine oxonols with moderate hydrophobicity tend to be sensitive fluorescent indicators for optical measurement of membrane potentials, and to be less prone to effects of extracellular environmental changes, e.g. culture medium and temperature etc. The substitutes on the nitrogen atoms of two thiobarbituric acid moieties need be critically fine-tuned. As a continuation-in-part to U.S. patent application Ser. No. 10/971,311 this invention provides an improved method for optical measurement of membrane potentials by using N,N,N′-trialkyl thiobarbituric acid-derived polymethine oxonols that have minimal serum effect compared to the existing N,N,N′,N′-tetraalkyl thiobarbituric acid-derived polymethine oxonols. The typical N,N,N′-trialkyl oxonols are generally prepared as shown in FIG. 1.